Urinary tract infections (UTIs) are among the most common bacterial infections, accounting for tremendous morbidity and healthcare costs. Up to 60% of women will experience a UTI in her lifetime, with between 25 and 40% of these women suffering frequent recurrences, recalcitrant to antibiotic therapy. More than 80% of community acquired and 50% of nosocomial UTIs are caused by uropathogenic E. coli (UPEC). The pathogenesis of UTI is complex with UPEC capable of colonizing multiple niches within the bladder and the kidneys. UPEC utilize surface expressed extracellular organelles known as type 1 pili to attach to, invade, and replicate within the terminally differentiated superficial facet clls in the bladder epithelium. UPEC escape the endocytic vacuole and replicate in cytoplasm into large biofilm-like masses called intracellular bacterial communities (IBCs). Within this environment, UPEC replicate rapidly and are protected from elements of the innate immune response as well as clearance by micturition. Furthermore, UPEC within IBCs are resistant to antibiotic therapy that is otherwise effective against the organisms grown in broth culture. Formation of IBCs is essential for bacterial colonization of the bladder in mouse models, and IBCs have been detected in urine from women suffering recurrent UTI. Understanding the pathogenesis of UPEC with regard to its ability to invade and replicate intracellularly is paramount to identifying targets for novel therapeutics that prevent bladder invasion and persistence. Recent data suggests that the formation of IBCs within the first 24 hours after experimental inoculation of bacteria into the murine urinary tract constitutes a robust population bottleneck restricting bacterial diversity progressing to later stages of infection. Furthermore, during this same time period, the induction of a robust immune response predisposes mice to experience persistent bacteriuria and chronic cystitis. This proposal explores the hypothesis that occupation of an intracellular niche during acute infection directly leads to an immune response predisposing the host to persistent bacteriuria and recurrent/chronic cystitis. Utilizing a well- characterized murine model of UTI, the molecular mechanisms accounting for the dramatic bottleneck during acute UTI, the direct relationship between acute pathogenic events and long-term UTI outcome, and the impact of sequential bladder inoculation on infection will be determined. The objectives of this research are to investigate the key events during acute UTI pathogenesis that impact infection outcome. Completion of this research will help to identify key events in pathogenesis that can be targeted with novel therapeutics to limit the morbidity of UTI.